The purpose of this project is to elucidate the molecular mechanisms by which intercellular adhesion complexes form and remodel in response to mechanical load. Recent evidence demonstrates that mechanically initiated signaling at cell-cell junctions is a fundamental aspect of cell and developmental biology. Aberrant assembly and remodeling of intercellular junctions has likewise emerged as a defining feature of diseases including metastatic cancers, cardiomyopathies, and skin barrier defects. However, at present very little is known about how the complex protein assemblies present at cell-cell contacts convert molecule-scale forces into biochemical signals, or how mechanical cues govern the complex junctional dynamics that typify multicellular tissues. Previous work from our collaboration showed that a complex of E-cadherin, ?-catenin, and ?E-catenin forms a minimal force-sensing unit at adherens junctions (AJs). Separate work suggests that ?E-catenin additionally plays a central role in organizing epithelial tissues based on its interactions with vinculin, Epithelial Protein Lost in Neoplasm (EPLIN), Zonula Occludens (ZO)-1, and afadin, all of which bind actin and recruit other scaffolding and signaling proteins. In Aim 1 we will test the hypothesis that force-sensitive, cooperative actin binding by ?E-catenin and vinculin leads to dramatic increases in actin affinity over a very small range in force. This idea, if correct, would explain how a four-protein system amplifies small changes in force into dramatic alterations in adhesion stability and downstream signal transduction. Further, we will perform the first detailed biochemical and biophysical characterization of the interaction of the cadherin-catenin complex with EPLIN, ZO-1, and afadin. These studies lay the foundation for a quantitative understanding for how the AJ functions as an integrated, multifunctional force-sensing assembly. In Aim 2 we will examine force sensitivity in desmosomes. These junctions link desmosomal cadherins to the intermediate filament (IF) cytoskeleton, and are essential for tissue integrity. However, while cel biological data suggest a role of desmosomes in transmitting force between cells, there is currently no direct evidence for when, where, and even whether desmosomal cadherins transmit tension between neighboring cells in the absence of externally applied force. To address this gap, we will use genetically encoded molecular tension sensors to determine when and where desmosomal cadherins transduce force between neighboring cells. We will then critically evaluate the role of desmoplakin in transmitting force at desmosomes, analogous to the role established for ?E-catenin at AJs. Finally, we will use a single-molecule magnetic tweezers assay to test the innovative hypothesis that recruitment of plakoglobin, plakophilin, or both to desmoplakin is inherently force sensitive. These experiments will dramatically enhance our basic understanding of how desmosomes function as a mechanical linkage between cells.